Abstract The enzyme 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2) converts 1-acylglycerol-3-phosphate (lysophosphatidic acid, LPA) to 1, 2- diacylglycerol-3-phosphate (phosphatidic acid, PA) in the triglyceride and glycerophospholipid biosynthesis pathways. Loss-of-function mutations in AGPAT2 result in an autosomal recessive form of congenital generalized lipodystrophy (CGL), which is characterized by a generalized lack of adipose tissue, insulin resistance, diabetes, hypertriglyceridemia and nonalcoholic fatty liver disease (NAFLD). NAFLD, insulin resistance and type 2 diabetes are burgeoning diseases typically associated with obesity; however, these diseases also occur in individuals who have a near total absence of adipose tissue, suggesting that they share important underlying causal mechanisms. Previously, we generated and characterized a mouse model of Agpat2 deficiency that recapitulates nearly all of the features of human CGL. Despite several studies in animal models and humans with CGL, the molecular mechanisms responsible for the loss of fat, insulin resistance and NAFLD remain unknown. Here, we will utilize our global AGPAT2 knockout mouse model in combination with a new tissue-specific mouse model in which we can inducibly inactivate or reactivate Agpat2 gene expression, to directly assess the underlying mechanisms responsible for the development of hepatic insulin resistance and steatosis, as well as address how the loss of AGPAT2 alters adipocyte development (Scherer). In Specific Aim 1, we will define the molecular mechanisms responsible for the development of hepatic steatosis and insulin resistance in mice that lack AGPAT2, by identifying the responsible transcription factor(s) and whether increased free fatty acid (FFA) flux to the liver, or accumulation of additional lipid species, such as ceramides, are responsible for the observed steatosis and hepatic insulin resistance. Specific Aim 2 will determine the requirement of AGPAT2 in hepatocytes for the development of hepatic steatosis, insulin resistance and hyperglycemia, through the characterization of mice that lack AGPAT2 only in hepatocytes and in mice that express AGPAT2 only in hepatocytes. Finally, Specific Aim 3 will elucidate how the absence of AGPAT2 leads to altered adipocyte differentiation, using a combination of in vitro and in vivo studies of mice, in which we can inducibly delete or re-express AGPAT2 selectively in adipocytes. Completion of these Aims will provide novel insights into the pathogenesis of insulin resistance and NAFLD, and will further, yield new targets and strategies for the diagnosis and treatment of these potentially life- threatening disorders. Similarly, the interrogation of mechanisms responsible for the lipodystrophy due to the loss of AGPAT2, will result in a more complete understanding of the basic mechanisms responsible for adipocyte differentiation.